Biological cells are encapsulated in a membrane made of a double layer of lipids separating the intracellular contents from the outside. The lipid bilayer “sandwich” has a hydrophobic interior that prevents movement of charged particles such as ions across it. However, there are protein macromolecules that penetrate the membrane and act as portholes to allow ions to pass between the inside and outside of a cell. These structures that allow rapid movements of ions (many millions per second) across a cell membrane, with no need for an immediate energy input, are called “ion channels”. The forces that influence the movement of ions through a channel are electrical and chemical. The electrical force is the electrical potential across the membrane, the chemical force is the difference in concentration of an ion on the two sides of the membrane: the combination of the two is the electrochemical gradient for an ion. If the electrochemical gradient for an ion is not zero, ions will flow through a channel when it opens (as long as the channel lets them through).
There are many varieties of ion channels that differ in their selectively, methods of gating, conductance and kinetic properties. Channels can be selective for sodium ions, or for potassium ions, or for calcium ions, or for chloride ions, or form protons etc and are classified according to the ions that pass through them most freely. For example, sodium channels are more permeable to sodium than to any other cations or anions. Channels are also classified according to the way in which they are turned on or gated. For example, voltage-activated channels open or close in response to changes in membrane potential. Ligand-gated channels are turned io when ligands such as neurotranmitters or hormones bind to their surface. Proteins to which ligands bind are commonly called receptors and many receptors are part of the same macromolecule that forms the ion channel. However, some channels are indirectly linked to receptors by second messenger systems and the channel is then separate from the receptor. Channels can also have very different conductances. Conductance, the reciprocal of resistance, is a measure of the ease with which ions pass through a channel and is given by the ratio of the current to the driving force. The conductance of different channels can range from picosiemens to hundreds of picosiemens (corresponding to resistances of 109 to 1012 ohms). Finally, channels can have very different “duty cycles”. Some are open most of the time while others open infrequently. Some flicker rapidly between open and closed states while others do not. Changes in the environment of channels (e.g the presence of drugs) can change these characteristics. Indeed it is becoming clear that many drugs exert their effects on cells and organs by binding to surface receptors and influencing channel behaviour.
The function of all cells in an animal or other organism depends on the ion channels formed by membrane proteins which provide a pathway for movement of ions between compartments in a cell and between the interior and exterior of cells. These movements of ions are essential for normal cell function, and all biological cells (including bacteria and even enveloped viruses such as the influenza and HIV viruses) contain ion channels. Ion channels are fundamental to cellular functions such as transmission of signals in nervous systems, cell division, production of antibodies by lymphocytes, replication of virus particles within cells and secretion of fluid and electrolytes.
A wide variety of diseases such as cystic fibrosis, muscular dystrophies, stroke, epilepsy and cardiac arrhythmias are related to disorders of ion channel function. In addition, it has recently been discovered that some viruses have proteins that form ion channels that are needed in the normal life-cycle of the virus. For example, there is now good evidence that a protein (M2) in influenza A virus forms an ion channel that is necessary for virus replication, and drugs such as amantadine that block this channel inhibit replication of the influenza A virus. Amantadine (1-aminoadamantane hydrochloride) and its analogue rimantidine have been found empirically to be effective in the prophylaxis and treatment of influenza caused by the influenza A virus. These drugs, at the therapeutic concentrations, inhibit replication of the influenza A virus both in vitro and in vivo. However, they can become ineffective because of the development of resistant strains of the virus and this reduces their value as therapeutic agents.
Other drugs which work by modulating ion channel function include calcium channel blockers which are used as anti-anginal and antihypertensive agents, barbiturates which cause sleep and inhibit epileptic seizures by increasing movements of chloride ions across receptors activated by gamma-amininobutyric acid (GABA), and benzodiazepines which relieve anxiety and produce anaesthesia by increasing GABA receptor activity.
In the past, the discovery of drugs which block ion channels has been largely serendipitous. Drugs that have been discovered in this way include general anaesthetics such as ether and halothane, the barbiturates and benzodiazepines. Thus, ether was originally used like alcohol at parties, and the reversible anaesthetic effect of halothane was discovered during leakage of refrigerant from a compressor. Similarly, the discovery of the antiarryhthmic action of quinidine followed use of quinine as an antimalarial drug.
Realisation that ion channels could prove to be an important site of drug action has lead to a search for effective ways of screening the activity of potential therapeutic substances that affect ion channel activity. Although electrophysiological techniques can be used to detect current flow when ions move across channels, the methods are too tedious and time-consuming for routine screening of ion channel activity.
Vpu is a small phosphorylated integral membrane protein encoded by the HIV-1 genome which associates with the Golgi and endoplasmic reticulum membranes in infected cells, but has not been detected in the plasma membrane nor in the viral envelope. The protein is 80-82 amino acids long depending on the viral isolate, with an N-terminal transmembrane anchor and a hydrophilic cytoplasmic C-terminal domain. The C-terminal domain contains a 12 amino-acid sequence that is conserved in all isolates and contains two serine residues that are phosphorylated. Using standard techniques associated with reconstitution of the purified HIV-1 Vpu protein in planar lipid bilayers, it has been shown that the Vpu protein forms cation selective ion channels in phospholipid bilayers (8). Further work is now directed to finding drugs that block these channels, and testing them as potential anti-HIV-1 therapeutic agents. While screening for such drugs is possible using the above mentioned planar lipid bilayer method, this method has the disadvantage of requiring large quantities of highly purified Vpu protein and is limited in that only one compound can be tested per bilayer, making it a relatively slow and inefficient screening assay.
Because of these disadvantages, there is a need for an ion channel assay system that can be used both to detect the ion channel activity of biologically important peptides and proteins, and to screen the effectiveness of potential therapeutic substances that might interact with ion channels and modulate ion channel function.
Some organisms such as bacteria accumulate amino acids and other substances by using the energy of a cation concentration gradient. If a substance such as a peptide, polypeptide or protein that forms a channel is inserted in the cell membrane and dissipates the gradient, the organism can no longer accumulate essential substances and growth is inhibited. This growth inhibition can be detected directly. Thus, the activity of potential therapeutic substances that might influence the function of a channel can be quickly screened by examining their effects on growth of an organism containing the channel-forming peptide, polypeptide or protein.